Many genetic diseases arise each generation because a parent transmits a brand new mutation to a child. Some mutations occur at frequencies 100-1,000 greater than what would be expected based on genome-average rates;many of these mutations are more likely to originate in the father than the mother, and to originate in older fathers than younger fathers (paternal age effect). Apert syndrome is an example of such a disease and is caused by mutations in the receptor tyrosine kinase (RTK) gene FGFR2. The high mutation frequencies could be due to greater than average mutation rates at the causal nucleotide sites, i.e. mutation "hot spots", or paradoxically, to positive selection on mutated premeiotic testis cells, whereby the mutation provides a growth advantage over the wild-type cells despite the consequences to the offspring. A method is available to distinguish between these two hypotheses by dissecting the testis of an unaffected male and measuring the mutation frequencies in each testis piece. If the mutation site is a hot spot, all testis pieces are expected to have elevated mutation frequencies. If germline selection is responsible, clusters with elevated mutation frequencies, surrounded by testis pieces with much lower mutation frequencies, are expected. For Apert syndrome mutations, in older testis donors, mutation clusters indicating germline selection were found. In younger testis donors, mutation clusters were not found, demonstrating that these clusters, and the mutation frequency, grew in the adult to contribute to the paternal age effect. The demonstration that an increase in disease mutation frequency might be due to germline selection is unprecedented. New disease mutations in different genes that show an unexpectedly high frequency will be studied using the testis dissection approach to distinguish between mutation hot spots or germline selection. First, disease mutations in different RTK genes will be analyzed to establish the relationship between the mutation frequency per germline cell division, the extent of any selective advantage and the magnitude of the observed paternal age effect. The second and third aims will examine disease mutations whose properties might reveal the altered protein functions and signal transduction pathways involved in providing the germline selective advantage. Finally, the fourth aim will develop improved methods for detecting very rare mutation events. PUBLIC HEALTH RELEVANCE: Using a new set of molecular tools we aim to understand the relative roles played by new mutation, paternal age and germline selection in establishing the unexpectedly high population burden of certain relatively well understood genetic diseases. Paternal age has become more recognized as a significant factor in societies where couples are delaying the time when they first have children. Learning about the factors responsible for the unexpectedly high mutation frequencies of the diseases studied in this grant may provide insight into the mechanisms behind the paternal age effect exhibited by less well understood diseases (e.g. autism and schizophrenia). Finally, developing new technologies for rare mutation detection could also be useful in cancer diagnostics and forensics as well as in discovery research.